Our laboratory has been studying factors that induce deamination in DNA. We have used model nucleic acid base and polymer systems to examine two inter-related hypotheses: (l) that ionized and protonated base pairs may be induced to form in DNA, and (2) that they may provide one pathway that can lead to induced deamination in DNA. We hypothesize that protonated base pairs may arise from several different kinds of DNA alterations, and that the deamination of protonated cytosines may comprise a previously undetermined and significant source of genetic mutations. In order to study induced deamination, we have developed a sensitive reversion assay by which the rates of deamination at a single site can be assessed under a variety of conditions. In the present grant, we propose to extend our studies in three ways. First, we will measure the rates of deamination of 5-methylcytosine (5mC) at 37 degrees C at specific locations in single (ss) and double-stranded (ds) DNA and compare the rates of deamination with those of unmethylated cytosine at the same site and under similar conditions. 5mC deaminates directly to thymine. Kinetic parameters for this reaction have never been measured under physiological conditions. There are three sites in our target where DNA can be methylated, and where we can measure the deamination of 5mC. Second, we have observed that CC-->TT tandem double mutations occur with unusually high frequency in double-stranded DNA incubated for long periods of time. The results are unusual in that the CC-->TT frequency is higher than even that of single C-->T mutants and the lesion presumably involves a uracil intermediate because tandem double mutations are nearly eliminated in an ung+ strain. The time dependent induction of double mutants may represent some type of induced mutation phenomena that has not previously been characterized. We will search for the cause(s) of tandem double mutants, determine their rates of induction by various agents, and identify the lesion(s) responsible. Third, we will complete our studies on the effect of the food additive, bisulfite ion, on the rate and mechanism of deamination of cytosine in double stranded DNA. This will be done at physiological temperatures and pH, under anaerobic conditions, and in the presence and absence of free- radical inhibitors.